This lethal mutation was generated by breeding an ENU mutagenized AE/J male to a female homozygous for In(1)1RK and leaden (ln) and heterozygous for the dominant lethal mutation splotch (Sp). The offspring were bred back to mice homozygous for In(1)1RK and leaden and heterozygous for splotch (In1Rk Sp ln/In1Rk + ln). All leaden offspring were known to derive from non-mutated chromosomes are were discarded. Because the splotch phenotype was a required marker all offspring not expressing the splotch phenotype were discarded. Because splotch homozygotes die in utero those expressing the splotch phenotype had the potential to carry an induced recessive lethal mutation and these splotch phenotype mice were then intercrossed or backcrossed to its parent. The presence of a recessive lethal mutation was evident in this G3 population because the splotch homozgyotes died in utero, those expressing splotch would only carry any recessive lethal mutation in repulsion (on the chromosome without the splotch allele), and those homozygous for any recessive lethal would die. Thus, if 100% of offspring or close to 100% of offspring expressed splotch then a recessive lethal mutation had been identified and captured in repulsion with splotch, leaden, and In(1)1Rk. This was how the l1Rk9 mutation was generated and identified (Roderick 1970).

Because the lethal mutation could only be identified by markers in repulsion this line was maintained by either intercrossing the l1Rk9/In1Rk Sp ln carriers or breeding a carrier to an In1Rk Sp ln/In1Rk + ln and selecting the splotch carriers that were not leaden as breeders. Embryos were generated for cryopreservation by breeding l1Rk9/In1Rk Sp ln carrier males to females that were either In1Rk Sp ln/In1Rk + ln or In1Rk + ln/In1Rk + ln. The recovered pups that express the splotch mutation are expected to carry the recessive lethal l1Rk9, although there is a chance of crossover such that 1 in 22 splotch expressing mice are statistically predicted to have lost the lethal mutation.